Carbon nanotube binding peptides

ABSTRACT

Peptides have been generated that have binding affinity to carbon nanostructures and particularly carbon nanotubes. Peptides of or the invention are generally about twelve amino acids in length. Methods for generating carbon nanotube binding peptides are also disclosed.

This application is a Divisional of granted U.S. patent application Ser.No. 11/352,582 filed Feb. 13, 2006, now granted U.S. Pat. No. 7,829,504,which under 35 U.S.C. §120 claims the benefit of U.S. patent applicationSer. No. 10/453,415—filed on Jun. 3, 2003—now U.S. Pat. No. 7,304,128granted Dec. 4, 2007.

FIELD OF THE INVENTION

The invention relates to methods, and compositions useful formanipulation, purification and characterization of carbon nanotubes.More specifically, the invention relates to peptides that bind carbonbased nanostructures, their synthesis and methods of use.

BACKGROUND OF THE INVENTION

Carbon nanotubes (CNT) have been the subject of intense research sincetheir discovery in 1991. CNT's possess unique properties such as smallsize and electrical conductivity, which makes them suitable in a widerange of applications, including use as structural materials inmolecular electronics, nanoelectronic components, and field emissiondisplays. Carbon nanotubes may be either multi-walled (MWNTs) orsingle-walled (SWNTs), and have diameters in the nanometer range.

Depending on their atomic structure CNT's may have either metallic orsemiconductor properties, and these properties, in combination withtheir small dimensions makes them particularly attractive for use infabrication of nano-devices.

One of the drawbacks to the implementation of CNT's in nano-devicefabrication processes is the difficulty in obtaining samples of CNT'sthat have uniform lengths, or chirality. Additionally, no facile methodis available for the immobilization and manipulation of CNT's fornano-device fabrication.

Most methods of CNT synthesis produce a product that is a mixture ofentangled tubes of “ropes”, giving CNT's differing in diameter,chirality, and in the number of walls. Various methods such as acidwashing, ultra-sonification, polymer wrapping and use of surfactantshave been employed for nanotube separation (J. Liu et al. Science 280,1253 (1998); A. G. Rinzler, Appl. Phys. 67, 29 (1998); A. C. Dillion etal. Adv. Mater. 11, 1354 (1999); (Schlittler et al. Science 292:1136(2001)).). However, there has been no report of a method for thespecific disentangling of nanotube ropes or their separation intopopulations having discrete sizes, chirality or conducting properties.

Because of their ability to specifically recognize substrates, variousproteins represent one possible route to solving the CNTseparation/purification problem as well as providing a possible meansfor CNT immobilization. Some attempts have been made to raise antibodiesto various carbon based structures. For example, Chen et al. (WO01/16155 A1) used conjugated fullerenes to raise monoclonal antibodiesto C₆₀ fullerene as a hapten. However, the population of antibodiesraised by immunization of mice with this C₆₀ fullerene derivative whichwas conjugated to bovine thyroglobulin included a sub-population thatcross reacted with a C₇₀ fullerene. No attempts have been made to dateto raise antibodies to carbon based nanotubes.

Since its introduction in 1985 phage display has been widely used todiscover a variety of ligands including peptides, proteins and smallmolecules for drug targets. (Dixit, S., J. of Sci. & Ind. Research, 57,173-183, 1998). The applications have expanded to other areas such asstudying protein folding, novel catalytic activities and DNA-bindingproteins with novel specificities. Whaley et al (Nature, 405:665 (2000))has used phage display technique to identify peptide sequences that canbind specifically to different crystallographic forms of inorganicsemiconductor substrates. Although the method of generating large,diverse peptide libraries with phage display has been known for sometime, it has not been applied to the problem of finding peptides thatmay be useful in the binding and manipulation of CNT's.

The problem to be solved, therefore, is to provide materials that havebinding specificity to CNT's and other carbon based nanostructures sothat they may be used in separation and immobilization of thesestructures for the fabrication of nano-devices. Applicants have solvedthe stated problem by providing a series of carbon nanotube bindingpeptides with high affinity and specificity for CNT's.

SUMMARY OF THE INVENTION

In one aspect the invention provides a process for generating a carbonnanostructure binding peptide comprising a composition comprising asolid substrate coated with a peptide having an amino acid sequenceselected from the group consisting of SEQ ID No: 167-177.

Provided herein are also compositions comprising a solid substratecoated with a carbon nanotube binding peptide, as well as compositionscomprising a solid substrate coated with a carbon nanotube bindingpeptide having at least one carbon nanotube bound thereto.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE LISTINGS

FIG. 1 is a TEM image of phages with carbon nanotube binding peptides onsurface of carbon nanotubes.

FIG. 2A is an electronmicrograph of untreated nanotubes ropes.

FIG. 2B is an electronmicrograph of single walled nanotubes treated withcarbon nanotube binding peptide as set forth in SEQ ID NO:13.

FIG. 2C is an electronmicrograph of single walled nanotubes treated witha control peptide, having little or no binding affinity for CNT's.

FIG. 3A is an electronmicrograph of a microsphere coated with a non-CNTbinding control phage after exposure to SWNT.

FIG. 3B is an electronmicrograph of a microsphere coated with aCNT-binding phage after exposure to SWNT.

FIG. 3C is an electronmicrograph of a microsphere coated with a non-CNTbinding peptide after exposure to SWNT.

FIG. 3D is an electronmicrograph of a microsphere coated with a CNTbinding peptide after exposure to SWNT.

The following sequences conform with 37 C.F.R. 1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) andconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R.§1.822.

SEQ ID Nos:1-24 and 35-39 are carbon nanotube binding peptides of theinvention.

SEQ ID NOs:25, and 32 are derivatized carbon nanotube binding peptideshaving a polyglycine tail.

SEQ ID Nos:26 and 27 are mutant carbon nanotube binding peptides havinga serine substituted in place of a tryptophan at position 6.

SEQ ID NO:28 is a control peptide that have little or no bindingaffinity for carbon nanostructures.

SEQ ID NO:29 is a charged portion of a carbon nanotube binding peptide.

SEQ ID NO:30 is a polar portion of a carbon nanotube binding peptide.

SEQ ID NO:31 is a hydrophobic portion of a carbon nanotube bindingpeptide.

SEQ ID NO:33 is a primer used for sequencing M13 phage.

SEQ ID NO:34 is a non-CNT binding peptide.

SEQ ID NOs:40-85 are peptides raised against and binding to singlewalled nanotoubes.

SEQ ID NOs:86-147 and 177 are peptides raised against and binding tomultiwalled carbon nanotubes.

SEQ ID NOs:148-176 are peptides raised against and binding to granphitecleaned carbon nanotubes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides various carbon nanotube binding peptidesgenerated by the process of peptide phage display. The peptides areuseful for the manipulation of carbon based nanostructures in thefabrication of nano-devices as well as in the separation andpurification of nanotubes from mixed CNT populations.

The peptides of the invention are particularly useful as ligands for theassembly of carbon nanotubes and related molecules into conducting nanodevices for use in electronic applications such as field-emissiontransistors, artificial actuators, molecular-filtration membranes,energy-absorbing materials, molecular transistors, and otheroptoelectronic devices as well as in gas storage, single-electrondevices, and chemical and biological sensors.

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided.

“CNBP” means Carbon nanotube binding peptide

“HRTEM” means high-resolution transmission electron microscopy

“MWNT” means Multi-walled nanotube

“SWNT” means Single walled nanotube

“PEG” means polyethylene glycol

“pfu” means plaque forming units

“TEM” means transmission electron microscopy

“CNT” means carbon nanotube

The term “peptide” refers to two or more amino acids joined to eachother by peptide bonds or modified peptide bonds. Peptides include thosemodified either by natural processes, such as processing and otherpost-translational modifications, but also chemical modificationtechniques. The modifications can occur anywhere in a peptide, includingthe peptide backbone, the amino acid side chain, and the amino orcarboxyl terminal. Examples of modifications include but are not limitedto amidation, acylation, acetylation, cross linking, cyclization,glycosylation, hydroxylation, phosphorylation, racemization, andcovalent attachment of various moieties such as nucleotide or nucleotidederivative, lipid or lipid derivatives (see, for instance,Proteins—Structure and Molecular Properties, 2^(nd) Ed Creighton, W.H.Freeman and Company, New York (1993) and Post-translation covalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York(1983)).

As used herein, the term “peptide” and “polypeptide” will be usedinterchangeably.

The term “nanotube” refers to a hollow article having a narrow dimension(diameter) of about 1-200 nm and a long dimension (length), where theratio of the long dimension to the narrow dimension, i.e., the aspectratio, is at least 5. In general, the aspect ratio is between 10 and2000.

By “carbon-based nanotubes” or “carbon nanotube” herein is meant hollowstructures composed primarily of carbon atoms. The carbon nanotube canbe doped with other elements, e.g., metals.

The term “carbon nanotube product” refers to cylindrical structures madeof rolled-up graphene sheet, either single-wall carbon nanotubes ormulti-wall carbon nanotubes.

The term “carbon nanotube rope” means a population of non-alignednanotubes.

The term “carbon nanostructure binding peptide” refers to peptides thatwere selected to bind with a carbon nanostructures. Where peptides aregenerated with specific affinity to carbon nanotubes, these peptideswill be referred to as carbon nanotube binding peptides or CNBP's.

The term “stringency” as it is applied to the selection of CNBP's meansthe concentration of eluting agent (usually detergent) used to elutepeptides from CNT's.

The term “peptide-nanotube complex” means structure comprising a peptidebound to a nanotube via a binding site on the peptide.

The term “nano-structure” means tubes, rods, cylinders, bundles, wafers,disks, sheets, plates, planes, cones, slivers, granules, ellipsoids,wedges, polymeric fibers, natural fibers, and other such objects whichhave at least one characteristic dimension less than about 100 nm.

The term “solid substrate” means a material to which a carbon nanotubeor binding peptide may be affixed either by direct chemical means or viaan intermediate material such as a coating.

The term “identity” refers to a relationship between two or morepolynucleotide sequences or two or more polypeptide sequences, asdetermined by comparing the sequences. “Identity” and “similarity” canbe readily calculated by known methods including but not limited tothose described in (Sequence Analysis in Molecular Biology, Von Heinje,G., Academic Press, 1987, Sequence analysis Primer, Gribskov, M. andDevereux, J., eds., M Stockton Press, New York, 1991, and ComputerAnalysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G.,eds, Humana Press, New Jersey, 1994.

The term “amino acid” will refer to the basic chemical structural unitof a protein or polypeptide. The following abbreviations will be usedherein to identify specific amino acids:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartic acid Asp D Asparagine oraspartic acid Asx B Cysteine Cys C Glutamine Gln Q Glutamine acid Glu EGlutamine or glutamic acid Glx Z Glycine Gly G Histidine His H LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV

The term “variant(s)” refers to a polynucleotide, or polypeptide, thatdiffers from a reference polynucleotide, or polypeptide, respectively,but retains essential properties. Changes in the nucleotide sequence ofthe variant may or may not alter the amino acid sequence of polypeptideencoded by the reference polynucleotide. Nucleotide changes may resultin amino acid substitutions, deletions, additions, fusions, andtruncations in the polypeptide encoded by the reference sequence. Atypical variant of a polypeptide may differ in amino acid sequence fromanother reference polypeptide by one or more substitutions, additions,deletions in any combinations. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such asallelic variant, or may not be known as naturally occurring variant.Non-naturally occurring variants of polynucleotides and polypeptides maybe made by direct synthesis, mutagenesis techniques, or by otherrecombinant methods known in the art.

The term “chemically equivalent amino acid” will refer to an amino acidthat may be substituted for another in a given protein without alteringthe chemical or functional nature of that protein. For example, it iswell known in the art that alterations in a gene which result in theproduction of a chemically equivalent amino acid at a given site, but donot effect the functional properties of the encoded protein are common.For the purposes of the present invention substitutions are defined asexchanges within one of the following five groups:

Hydrophobic

-   -   Small aliphatic, nonpolar or slightly polar residues: Ala, Ser,        Thr Pro, Gly;    -   Large aliphatic, nonpolar residues: Met, Leu, Ile, Val Cys; and    -   Large aromatic residues: Phe, Tyr, Trp;        Hydrophilic:    -   Polar, negatively charged residues and their amides: Asp, Asn,        Glu, Gln;    -   Polar, positively charged residues: His, Arg, Lys;        Thus, alanine, a hydrophobic amino acid, may be substituted by        another less hydrophobic residue (such as glycine) or a more        hydrophobic residue (such as valine, leucine, or isoleucine).        Similarly, changes which result in substitution of one        negatively charged residue for another (such as aspartic acid        for glutamic acid) or one positively charged residue for another        (such as lysine for arginine) can also be expected to produce a        functionally equivalent product. Additionally, in many cases,        alterations of the N-terminal and C-terminal portions of the        protein molecule would also not be expected to alter the        activity of the protein.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. A “foreign” gene refers to a gene not normally found in thehost organism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments which are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Suitable regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing site, effector binding site andstem-loop structure.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The term “host cell” refers to cell which has been transformed ortransfected, or is capable of transformation or transfection by anexogenous polynucleotide sequence.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

The term “phage” or “bacteriophage” refers to a virus that infectsbacteria. Altered forms may be used for the purpose of the presentinvention. The preferred bacteriophages are derived from two “wild”phages, called M13 and lambda. Lambda phages are used to clone segmentsof DNA in the range of around 10-20 kb. They are lytic phages, i.e.,they replicate by lysing their host cell and releasing more phages. TheM13 system can grow inside a bacterium, so that it does not destroy thecell it infects but causes it to make new phages continuously. It is asingle-stranded DNA phage.

The term “phage display” refers to the display of functional foreignpeptides or small proteins on the surface of bacteriophage or phagemidparticles. Genetically engineered phage could be used to presentpeptides as segments of their native surface proteins. Peptide librariesmay be produced by populations of phage with different gene sequences.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook, J., Fritsch, E. F.and Maniatis, T., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L.and Enquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory Cold Press Spring Harbor, N.Y. (1984); and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-Interscience (1987).

The present invention provides peptides that bind carbon nanostructuresas well as methods for generating the same and uses thereof.

Carbon Nanostructures

The invention relates to the generation of peptides with bindingaffinities for carbon nanostructures, and particularly nanotubes. Carbonnano-structures of the present invention are those structures comprisedat primarily of carbon which take the form of tubes, rods, cylinders,bundles, wafers, disks, sheets, plates, planes, cones, slivers,granules, ellipsoids, wedges, polymeric fibers, natural fibers, andother such objects which have at least one characteristic dimension lessthan about 100 nm. Preferred carbon nanostructures of the invention arenanotubes.

Nanotubes of the invention are generally about 1-200 nm in diameterwhere the ratio of the length dimension to the narrow dimension, i.e.,the aspect ratio, is at least 5. In general, the aspect ratio is between10 and 2000. Carbon nanotubes are comprised primarily of carbon atoms,however may be doped with other elements, e.g., metals. The carbon-basednanotubes of the invention can be either multi-walled nanotubes (MWNTs)or single-walled nanotubes (SWNTs). A MWNT, for example, includesseveral concentric nanotubes each having a different diameter. Thus, thesmallest diameter tube is encapsulated by a larger diameter tube, whichin turn, is encapsulated by another larger diameter nanotube. A SWNT, onthe other hand, includes only one nanotube.

Carbon nanotubes (CNT) may be produced by a variety of methods, and areadditionally commercially available. Methods of CNT synthesis includelaser vaporization of graphite (A. Thess et al. Science 273, 483(1996)), arc discharge (C. Journet et al., Nature 388, 756 (1997)) andHiPCo (high pressure carbon monoxide) process (P. Nikolaev et al. Chem.Phys. Lett. 313, 91-97 (1999)). Chemical vapor deposition (CVD) can alsobe used in producing carbon nanotubes (J. Kong et al. Chem. Phys. Lett.292, 567-574 (1998); J. Kong et al. Nature 395, 878-879 (1998); A.Cassell et al. J. Phys. Chem. 103, 6484-6492 (1999); H. Dai et al. J.Phys. Chem. 103, 11246-11255 (1999)).

Additionally CNT's may be grown via catalytic processes both in solutionand on solid substrates (Yan Li, et al., Chem. Mater.; 2001; 13(3);1008-1014); (N. Franklin and H. Dai Adv. Mater. 12, 890 (2000); A.Cassell et al. J. Am. Chem. Soc. 121, 7975-7976 (1999)).

Peptide Generation

Peptides of the invention are generated randomly and then selectedagainst a population of carbon nanostructures for binding affinity toCNT's. The generation of random libraries of libraries of peptides iswell known and may be accomplished by a variety of techniques including,bacterial display (Kemp, D. J.; Proc. Natl. Acad. Sci. USA 78(7):4520-4524, 1981, and Helfman, D. M., et al., Proc. Natl. Acad. Sci. USA80(1): 31-35, 1983) yeast display (Chien C T, et al., Proc Natl Acad SciUSA 1991 Nov. 1; 88(21): 9578-82) combinatorial solid phase peptidesynthesis (U.S. Pat. Nos. 5,449,754, 5,480,971, 5,585,275, 5,639,603)and phage display technology (U.S. Pat. Nos. 5,223,409; 5,403,484;5,571,698; 5,837,500). Techniques to generate such biological peptidelibraries are described in Dani, M., J. of Receptor & SignalTransduction Res., 21(4), 447-468 (2001).

A preferred method to randomly generate peptides is by phage display.Phage display is an in vitro selection technique in which a peptide orprotein is genetically fused to a coat protein of a bacteriophage,resulting in display of fused protein on the exterior of phage viron,while the DNA encoding the fusion residues within the virion. Thisphysical linkage between the displayed protein and the DNA encoding itallows screening of vast numbers of variants of proteins, each linked toa corresponding DNA sequence, by a simple in vitro selection procedurecalled “biopanning.” In its simplest form, biopanning is carried out byincubating the pool of phage-displayed variants with a target ofinterest that has been immobilized on a plate or bead, washing awayunbound phage, and eluting specifically bound phage by disrupting thebinding interactions between the phage and target. The eluted phage isthen amplified in vivo and the process repeated, resulting in stepwiseenrichment of the phage pool in favor of the tightest binding sequences.After 3 or more rounds of selection/amplification, individual clones arecharacterized by DNA sequencing.

Thus it is an object of the invention to provide a process forgenerating a carbon nanostructure binding peptide comprising:

-   -   a) providing a library of phages expressing peptides in        solution;    -   b) providing a population of carbon nanostructures;    -   c) contacting the phage of (a) with the nanostructures of (b)        for a time sufficient to permit binding of the phage to the        nanostructures and forming a phage-nanostructure complex;    -   d) removing unbound phage;    -   e) contacting the phage-nanostructure complex of (c) with a        suitable bacterial host whereby the bacteria are infected by the        phage;    -   f) growing the infected bacteria of step (e) for a time        sufficient to permit replication of the phage and the expressed        peptide; and    -   g) isolating the replicated phage and expressed peptide of        step (f) wherein the peptide binds carbon nanostructures.        Peptide Selection

After a suitable library of peptides has been generated they are thencontacted with an appropriate population of carbon nanostructures ornanotubes. The nanotubes are presented to the library of peptidestypically while suspended in solution, although it will be appreciatedthat CNT or peptides could also be immobilized on a solid substrate tofacilitate binding. In such an embodiment suitable solid substrates willinclude but are not limited to silicon wafers, synthetic polymersubstrates, such as polystyrene, polypropylene,polyglycidylmethacrylate, substituted polystyrene (e.g., aminated orcarboxylated polystyrene; polyacrylamides; polyamides;polyvinylchlorides, etc.); glass, agarose, nitrocellulose, and nylon.

A preferred solution is a buffered aqueous saline solution containing asurfactant. A suitable solution is Tris-buffered saline with 0.1% Tween20. The solution can additionally be agitated by any means in order toincrease binding of the peptides to the nanotubes.

Upon contact a number of the randomly generated peptides will bind tothe CNT's to form a peptide-nanotube complex. Unbound peptide and CNTmay be removed by washing (if immobilized) or by any other means such ascentrifugation, or filtering, etc. After all unbound material isremoved, peptides, having varying degrees of binding affinities forCNT's may be fractionated by selected washings in buffers having varyingstrengths of surfactants. The higher the concentration of surfactant inthe wash buffer, the higher the stringency of selection. Increasing thestringency used will increase the required strength of the bond betweenthe peptide and nanotube in the peptide-nanotube complex.

A number of materials may be used to vary the stringency of the buffersolution in peptide selection including but not limited to acidic pH1.5-3; basic pH 10-12.5; high salt concentrations such as MgCl2 3-5 M,LiCl 5-10 M; water; ethylene glycol 25-50%; dioxane 5-20%; thiocyanate1-5 M; guanidine 2-5 M; urea 2-8 M; various concentrations of differentsurfactants such as SDS (sodium dodecyl sulfate), DOC (sodiumdeoxycholate), Nonidet P-40, Triton X-100, Tween 20® wherein Tween 20®is preferred. The materials can be prepared in buffer solutionsincluding but not limited to Tris-HCl, Tris-borate, Tris-acidic acid,triethylamine, phosphate buffer, glycine-HCl wherein 0.25M glycine-HClsolution is preferred.

It will be appreciated that peptides having greater and greater bindingaffinities for the CNT substrate may be eluted by repeating theselection process using buffers with increasing stringencies.

The eluted peptides can be identified, sequenced, and produced by anymeans known in the art.

Carbon Nanotube Binding Peptides

Peptides of the invention selected by the above process have beenidentified. A large number of peptides having particularly high bindingaffinities to carbon nanotubes were isolated having the amino acidsequences as set forth in SEQ ID NOs:1-24 and 35-177

It will be appreciated by the skilled artisan that the invention is notlimited to these specific sequences but will include amino acidsequences comprising chemically equivalent amino acid substitutions thatdo not interfere with the ability of the peptide to bind CNT's. So forexample, the chemically equivalent substitutions for each of the aminoacids in SEQ ID NO:14 are detailed in the following table:

SEQ ID NO: 14 His Trp Ser Ala Trp Trp Ile Arg Ser Asn Gln Ser EquivalentLys Phe Pro Ser Phe Phe Lys Pro Asp Asp Pro Amino Acids Arg Tyr Ala ProTyr Tyr His Ala Glu Asn Ala Thr Thr Thr Gln Glu Thr Gly Gly Gly Gly

Alignment and analysis of the selected peptides of the inventionsuggests that the carbon nanostructure or nanotube binding propertiesare related to the secondary characteristics of the peptide. For examplea simple pendant model was developed for the peptides of the instantinvention, which accounts for hydrophilicity or hydrophobicity. Itdemonstrates that all of the consensus sequences are essentiallysymmetric surfactants—hydrophilic on the ends and hydrophobic in themiddle. The model describes the degree of hydrophilicity orhydrophobicity of an amino acid pendant group by classifying all pendantgroups as either hydrophilic (h=−1) or hydrophobic (h=1). Side chainswhich are either basic, acidic or uncharged polar are be hydrophilicwhile side chains that are nonpolar are hydrophobic. Several of thepeptides selected by the methods of the invention are modeled below:

(SEQ ID NO: 1) H A H S Q W W H L P Y R −1 1 −1 −1 −1 1 1 −1 1  1 −1 −1(SEQ ID NO: 13) H W K H P W G A W D T L −1 1 −1 −1  1 1 1  1 1 −1 −1  1(SEQ ID NO: 14) H W S A W W I R S N Q S −1 1 −1  1  1 1 1 −1 −1  −1 −1−1 (SEQ ID NO: 8) H N W Y H W W M P H N T −1 −1   1 −1 −1 1 1  1 1 −1 −1−1These peptides were selected over a broad range of detergentconcentrations (0.6%-3%) and yet show the same pattern of hydrophilicityand hydrophobicity. With a few exceptions, the h=−1 are predominantly onthe ends and h=1 are concentrated in the middle.

It is thus an object of the invention to provide a carbon nanotubebinding peptides having the general structure:N-M-CWherein:

N is the N-terminal portion of the peptide having about 4 amino acids,75% of which are hydrophilic;

M is the median portion of the peptide having about 4 amino acids, 75%of which are hydrophobic; and

C is the C-terminal portion of the peptide having about 4 amino acids75% of which are hydrophilic.

Peptide Production by Recombinant Methods

Once a peptide having suitable binding properties is identified it maybe produced recombinantly in large quantities. Genes encoding nanotubebinding peptides may be produced in heterologous host cells,particularly in the cells of microbial hosts.

Preferred heterologous host cells for expression of nanotube bindingpeptides are microbial hosts that can be found broadly within the fungalor bacterial families and which grow over a wide range of temperature,pH values, and solvent tolerances. Because of transcription, translationand the protein biosynthetic apparatus is the same irrespective of thecellular feedstock, functional genes are expressed irrespective ofcarbon feedstock used to generate cellular biomass. Examples of hoststrains include but are not limited to fungal or yeast species such asAspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, orbacterial species such as Salmonella, Bacillus, Acinetobacter,Rhodococcus, Streptomyces, Escherichia, Pseudomonas, Methylomonas,Methylobacter, Alcaligenes, Synechocystis, Anabaena, Thiobacillus,Methanobacterium and Klebsiella.

A variety of expression systems can be used to produce the peptides ofthe present invention. Such vectors include but are not limited tochromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, frominsertion elements, from yeast episoms, from viruses such asbaculaviruses, retroviruses and vectors derived from combinationsthereof such as those derived from plasmid and bacteriophage geneticelements, such as cosmids and phagemids. The expression systemconstructs may contain regulatory regions that regulate as well asengender expression. In general, any system or vector suitable tomaintain, propagate or express polynucleotide or polypeptide in a hostcell may be used for expression in this regard. Microbial expressionsystems and expression vectors contain regulatory sequences that directhigh level expression of foreign proteins relative to the growth of thehost cell. Regulatory sequences are well known to those skilled in theart and examples include but are not limited to those which cause theexpression of a gene to be turned on or off in response to a chemical orphysical stimulus, including the presence of a regulatory elements mayalso be present in the vector, for example, enhancer sequences. Any ofthese could be used to construct chimeric genes for production of theany of the nanotube binding peptides. These chimeric genes could then beintroduced into appropriate microorganisms via transformation to providehigh level expression of the peptides.

Vectors or cassettes useful for the transformation of suitable hostcells are well known in the art. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene, one or more selectable markers, and sequences allowingautonomous replication or chromosomal integration. Suitable vectorscomprise a region 5′ of the gene which harbors transcriptionalinitiation controls and a region 3′ of the DNA fragment which controlstranscriptional termination. It is most preferred when both controlregions are derived from genes homologous to the transformed host cell,although it is to be understood that such control regions need not bederived from the genes native to the specific species chosen as aproduction host. Selectable marker genes provide a phenotypic trait forselection of the transformed host cells such as tetracyclin orampicillin resistance in E. coli.

The gene can be placed under the control of a promoter, ribosome bindingsite (for bacterial expression) and, optionally, an operator or controlelement, so that Initiation control regions or promoters, which areuseful to drive expression of the instant ORF's in the desired host cellare numerous and familiar to those skilled in the art. Virtually anypromoter capable of driving these genes is suitable for the presentinvention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1,PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful forexpression in Saccharomyces); AOX1 (useful for expression in Pichia);and lac, ara, tet, trp, IP_(L), IP_(R), T7, tac, and trc (useful forexpression in Escherichia coli) as well as the amy, apr, npr promotersand various phage promoters useful for expression in Bacillus.

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary, however, it is most preferred if included.

The vector containing the appropriate DNA sequence as here in abovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the peptide of the present invention. Cell-free translationsystems can also be employed to produce such peptides using RNAs derivedfrom the DNA constructs of the present invention.

Optionally it may be desired to produce the instant gene product as asecretion product of the transformed host. Secretion of desired proteinsinto the growth media has the advantages of simplified and less costlypurification procedures. It is well known in the art that secretionsignal sequences are often useful in facilitating the active transportof expressible proteins across cell membranes. The creation of atransformed host capable of secretion may be accomplished by theincorporation of a DNA sequence that codes for a secretion signal whichis functional in the host production host. Methods for choosingappropriate signal sequences are well known in the art (see for exampleEP 546049; WO 9324631). The secretion signal DNA or facilitator may belocated between the expression-controlling DNA and the instant gene orgene fragment, and in the same reading frame with the latter.

Nano-Device Fabrication

The carbon nanotube binding peptides (CNBP) of the instant inventioncould be one element in an entity with bi-, tri- (or higher) bindingfunctionality. A CNBP can be depicted graphically as shown below,

where the “C” is suggestive of a carbon nanotube and a bindingfunctionality depicted by “⊂”. However, despite its being drawn at oneend, it should be interpreted as a collective, not localized, propertyof the peptide sequence. The overall entity would be constructed by“fusion” of the CNBP with another body, depicted by “B”, with a bindingfunctionality depicted by “>”, and the combination is representedgraphically below

This is meant to represent a minimal example; the fusion could createhigher order (enumerative) functionality. Examples of B include but arenot limited to a DNA binding protein, a metallic electrode, for exampleAu bound directly to an amino-acid residue like cysteine, or a hard(e.g., Si or SiO₂) substrate for immobilization of the CNBP.

Directed self-assembly of carbon nanotubes into useful structures couldbe achieved by combining the binding of CNBP with a pre-patternedsubstrate. For example, if the binding functionality “B” was a series ofcysteine residues, the sequence of (a) preparation of a dilutesuspension of carbon nanotubes, (b) functionalization of selected typesby CNBP, and (c) washing over a substrate with patterned Au electrodeswould result in the attachment of carbon nanotubes to metal electrodesvia the peptide, within distances of relevance to nano-electronicdevices. Because of the diversity of the bio-chemical toolkit incombining elements to obtain higher order functionality, many other suchmethods can be conceived, once the fundamental binding motifs have beenidentified.

A major obstacle to the use of carbon nanotubes in a variety ofapplications is the fact that all manufacturing processes produce amixture of entangled tubes. Individual tubes in the product differ indiameter, chirality, and number of walls. Moreover, long tubes show astrong tendency to aggregate into “ropes”. These ropes are formed due tothe large surface areas of nanotubes and can contain tens to hundreds ofnanotubes in one rope. Furthermore, the structure of individual tubesvaries widely from armchair, zig-zag or other chiral forms which coexistin the material and their electrical properties also vary dramaticallyaccordingly (metallic or semi-conductive). Therefore, a need exists forthe isolation of a single form (such as armchair, zig-zag or a chiralform) of carbon nanotubes.

Existing methods for separating such product, for example acid washing,ultra-sonification, and use of surfactants, is non-specific with respectto the type of nanotube. Because peptide binding is usually highlyspecific, a major utility of a CNBP is to effect specific separation.One possible method would use dilute suspensions of carbon nanotubesseparated by having them flow over substrates patterned with differenttypes of binding CNBP's. One would choose to order the patterning basedon strength and specificity of binding, i.e., strongly selective bindingpeptides would be positioned to act on the mixture in advance of lessspecific ones. Many other ways to achieve separation can be conceived.If “B” binds to a magnetic particle, the joint entity could be used inone stage of a continuous flow to bind to carbon nanotubes, while inanother stage the bound nanotubes could be separated magnetically.

Thus it is an object of the invention to provide a method of dispersinga population of carbon nanotube ropes comprising:

a) providing a population of carbon nanotubes in solution in ropeformation; and

b) contacting the population of carbon nanotubes of step (a) with acarbon nanotube binding peptide having the general structure:N-M-C

wherein:

N is the N-terminal portion of the peptide having about 4 amino acids,75% of which are hydrophilic;

M is the median portion of the peptide having about 4 amino acids, 75%of which are hydrophobic; and

C is the C-terminal portion of the peptide having about 4 amino acids75% of which are hydrophilic;

whereby the carbon nanotube ropes are dispersed.

One of skill in the art will appreciate that it will be useful to sortpopulations of nanotubes to select for various binding properties. It iscontemplated that CNT-binding peptides or isolated phage expressing aCNT binding peptide, may be used for this purpose. For example,CNT-binding peptides or phages expressing the same, having an affinityto a specific population of CNT's may be immobilized on a solidsubstrate and then contacted with a mixed population of CNT's. Thedesired CNT's will bind to the immobilized peptides or phage and theundesired CNT's may be washed free. Alternatively, solid substrates suchas beads or microspheres may be coated with CNT-binding peptides orphage and used to assemble CNT's. Materials suitable as solid supportsmay be made of synthetic polymers such as polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), nylon, poly(vinyl butyrate), or other materials such asglass, ceramics, metals, and the like. These materials be used as films,microtiter plates, wells, beads, slides, particles, pins, pegs, ormembranes.

Accordingly the invention a method for assembling carbon nanotubescomprising contacting a solid substrate coated with at least one speciesof carbon nanotube binding peptide with a population of carbon nanotubeswhereby the carbon nanotubes bind to the coated substrate and areassembled.

It will be appreciated by the skilled artisan that patterning ofCNT-binding peptides on a particular solid support will be a usefultechnique in the design and fabrication of nanodevices. In someinstances patterning may be achieved by partially and selectivelymasking portions of the support with materials that repel or have noaffinity for CNT's. A variety of materials may be used for this purpose,however non-CNT binding peptides of similar physical characteristics toCNT-binding peptides will be particularly suitable. Non-CNT bindingpeptides are easily selected and identified in the early rounds of anyselection process for CNT-binding peptides. These may be used to mask asolid support to effect the pattering of CNT binding on the support.Several examples provided herein include the peptides set forth in SEQID NO:28 and 34.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989)(Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, pub. by Greene Publishing Assoc. andWiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989). All reagents, restriction enzymes andmaterials used for the growth and maintenance of bacterial cells wereobtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories(Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma ChemicalCompany (St. Louis, Mo.) unless otherwise specified.

Preparation of Phage Library

The phage library used in the present invention was purchased from NewEngland BioLabs (catalog number E8110S, Ph.D.—12 Phage Display PeptideLibrary Kit). The kit is based on a combinatorial library of randompeptide 12-mers fused to a minor coat protein (plll) of M13 phage. Thedisplayed peptide 12mer is expressed at the N-terminus of pill, i.e.after the signal peptide is cleaved the first residue of the coatprotein is the first residue of the displayed peptide. The librarycontains 2.7×10⁹ (100 μl) variants in the displayed epitope. A volume of10 μl contains about 55 copies of each peptide sequence. To avoidintroduce bias into the library, each initial round of experiments werecarried out using the original library provided by the manufacture.

Sequencing of Phages

Random M13 phage plaques were picked and single plaque lysates wereprepared following manufacture's instruction (New England Labs, Beverly,Mass.). The single stranded phage genome DNA was purified with Qiagenekit (QIAprep Spin M13 kit, Cat. No. 27704). The single stranded DNA weresequenced with −96 glll sequencing primer (5′-CCCTCATAGTTAGCGTAACG-3′).SEQ ID NO:33 The displayed peptide is located immediately after thesignal peptide of gene III.

Multi-Sequence Analysis

Sequences from the phage display experiment were analyzed using softwareDNA Star (version 5.02) or ClustalW according to software instructions.The alignment provides similarities among the selected sequences andpredict important functions of certain amino acid residues.

Example 1 Preparation of Carbon Nanotubes

Carbon nanotubes designated CNT-7 were obtained from Yet-Ming Chiang,Department of Materials Science, MIT, Cambridge, Mass. The nanotubeswere prepared by heating SiC (silicon carbide) at 1700° C. under vacuum.Silicon “evaporated” from the sample and left behind the carbon, whichformed folded structures of carbon including nanotubes.

Single-walled carbon nanotubes were purchased from CNI (CarbonNanotechnology Incorporated, Houston, Tex.). These nanotubes wereproduced by a laser oven technique or a HiPCo (high-pressure carbonmonoxide) process (P. Nikolaev et al. Chem. Phys. Lett. 313, 91-97(1999)).

The CNT-7 sample contained various carbon structures includingmulti-wall and single-wall carbon nanotubes whereas CNI samples weremostly single wall carbon nanotubes.

Example 2 Selection of Carbon Nanotube Binding Peptides

CNT-7 and CNI carbon nanotubes were suspended in Tris-Buffered-Salinewith 0.1% Tween 20 (TBS-T) at a concentration of 1 mg/ml. The carbonnanotube solution was then sonicated by a Branson Sonifier model 450(Branson Sonic Power Co., Danbury, Conn.) with power output settingbetween 4 and 5, duty cycle 70-80% for three times. Ten microliters ofM13 phage library (containing about 10¹¹ phage) were added to 1 ml ofcarbon nanotubes. The mixture was incubated at room temperature withmild agitation for 60 minutes. Unbound phages were separated from thenanotube sample by high speed spin at 14,000 rpm (16,110×g) in anEppendorf 5415C centrifuge (Brinkmann Instruments Inc., Westbury, N.Y.)for 10 minutes. Subsequently the phage/nanotube complex was washed 10times each with 1 ml TBS-T in which concentration of Tween-20 increasesaccording to the cycles of selection, as shown in the data below. Forexample, in one experiment the Tween 20 concentration was increased from0.2% in round one, to 0.3% in round two, to 0.4% in round 4, to 0.5% inround 5, to 0.6% in round 6, to 0.7% in round 7, to 1% in round 8, to 2%in round 9, 3% in round 10, 6% in round 11 and 10% in round 12. Afterthe last (tenth) washing step, the bound phages were eluted off byincubating with 0.5 ml of 0.25 M glycine-HCl, pH 3.0 for 10-15 minutesat room temperature. The phages and nanotubes were separated bycentrifuging at 14,000 rpm (16,110×g) for 10 minutes, with the clearedsupernatant containing the eluted phages. The presence and concentrationof phages in the supernatant were determined by phage titering. Once thesample confirmed the presence of phages, they were used to inoculate E.coli for phage amplification, and the amplified phage sample was used asthe “pool” for next round. In a typical experiment, the entire eluentwas added to a 20 ml E. coli culture at early log phase. The culture wasfurther incubated for 4.5 hours at 37° C. to allow phage to propagate.At the end of the incubation, the cultures were spin at 16,000×g for 10minutes at 4° C. The phages in the cleared supernatant were precipitatedwith PEG/NaCl at 4° C. After centrifugation, the phages were resuspendedin 200 μl PBS and the concentration was determined by titering. Thissample is used subsequently as the stock for the next round experiment.To carry out the next round experiment, 10¹¹ phages were used as input“pool” and the selection process was repeated as described above withincreased stringency for washing, i.e. increased concentration ofTween-20. Useful peptides were obtained by selection at detergentconcentrations of 0.5% and higher and the amino acid sequences of thesepeptides are shown in Table 1.

TABLE 1 M13 peptide sequences for CNT-7 SEQ ID NO: Sequence 1.DPHHHWYHMHQH 2. HAHSQWWHLPYR 3. HAHSRRGHIQHR 4. HCHHPWGAWHTL 5.HCWNQWCSRHQT 6. HGNWSYWWSKPS 7. HHWHHWCMPHKT 8. HNWYHWWMPHNT 9.HNWYRWCIRHNN 10. HRWYRWSSRNQT 11. HSSWWLALAKPT 12. HWCAWWISSNQS 13.HWKHPWGAWDTL 14. HWSAWWIRSNQS 15. HWSPWHRPWYQP 16. HYSWYSTWWPPV 17.HYWWRWWMPNQT 18. KCHSRHDHIHHH 19. KSLSRHDHIHHH 20. KSRSRHDEIHHH 21.KYRSRHDHIHHH 22. QWHSRHDHIHHH 35. HNWYHWWPHNT 36. HWYKPYHFQSLT 37.SVSVGMKPSPRP 38. EAHPQTLGWQRP 39. HNAYWHWPPSMT

Binding of these peptides to CNT's is further confirmed by TEMmicrographs as shown in FIG. 1, illustrating a number of the subjectpeptides bound to a single walled CNT.

Example 3 Structural and Functional Characterization of Nanotube BindingPeptides

The following example illustrates the importance of conserved aminoacids to the binding affinity of peptides for carbon nanotubes.

Alignment of the selected peptide sequences suggests strongly thathistidine at position 1 and tryptophan at position 6 are important forbinding. Further analysis of more than one hundred phage clones, shownbelow in Table 3, revealed that His and Trp are two dominant amino acidsin the composition of peptides selected by the display.

TABLE 3 Change Number % by % by Original relative to Amino Acids countweight frequency library (%) original (%) Charged: 391 36.03 33.59RKHYCDE (SEQ ID NO: 29) Acidic: 37 2.84 3.18 DE Basic: 89 8.51 7.65 KRPolar: 305 22.51 26.20 NCQSTY (SEQ ID NO: 30) Hydrophobic: 376 37.6232.3 AILFW (SEQ ID NO: 31) A 57 2.69 4.90 6.0 — C 6 0.41 0.52 0.5 0.52 D35 2.67 3.01 2.8 3.01 E 2 0.17 0.17 3.1 0.17 F 5 0.49 0.43 3.3 0.43 G 301.13 2.58 2.6 2.58 H 217 19.73 18.64 6.3 — I 27 2.03 2.32 3.4 2.32 K 383.23 3.26 2.8 3.26 L 55 4.13 4.73 9.3 4.73 M 25 2.17 2.15 2.6 2.15 N 664.99 5.67 4.6 5.67 P 85 5.47 7.30 12.2 7.30 Q 37 3.14 3.18 5.1 3.18 R 515.28 4.38 4.7 4.38 S 97 5.60 8.33 10.0 8.33 T 57 3.82 4.90 11.1 4.9  V 60.39 0.52 3.9 0.52 W 226 27.90 19.42 2.2 — Y 42 4.54 3.61 3.6 3.61 B 0 00 Z 0 0 0 X 0 0 0 Ter 0 0 0 1. Data is from analysis of 100 clones 2.Original library data is adapted from manufacturer's manual and is fromanalysis of 104 clones

Site-directed mutagenesis was used to introduce mutations in peptidesSEQ ID NO:13 (HWKHPWGAWDTL) and SEQ ID NO:14 (HWSAWWIRSNQS) Trp->Ser atposition 6, to produce peptides HWKHPSGAWDTL (SEQ ID NO:26) andHWSAWSIRSNQS (SEQ ID NO:27), respectively. The phages carrying thesemutations were assayed for their binding activity against CNT-7 atdetergent concentration 0.4% as described above. The mutation at Trp6reduced binding to CNT-7 for both peptides. The data is shown in Table 4below. The number of plaque forming units is charted for a controlpeptide (LPPSNASVADYS) SEQ ID NO:28 and peptides SEQ ID NO:13, 14 andmutant peptides SEQ ID NO: 26 and 27. The binding data shown in Table 4confirms the critical role of Trp in binding to nanotubes.

TABLE 4 Phage Pfu SEQ ID NO: 26 4.58 × 10⁶ SEQ ID NO: 27  5.4 × 10⁶ SEQID NO: 28  6.7 × 10⁶ SEQ ID NO: 13 15.9 × 10⁶ SEQ ID NO: 14 59.2 × 10⁶

Example 4 Effect of Peptide Binding on Populations of Nanotubes

The following example illustrates the ability of carbon nanotube bindingpeptides to disentangle carbon nanotube “ropes”.

Experiments were carried out with synthetic peptides and single-wallcarbon nanotubes (CNI/Laser oven) and binding peptides sequenceHWKHPWGAWDTLGGG [SEQ ID NO: 25]. This peptide was selected as describedin Example 2 and represents the peptide as set forth in SEQ ID NO:13,with the addition of a poly-glycine tail.

At a concentration of 4 mg/ml peptide of SEQ ID NO:25 was seen todisperse the nanotube ropes as examined by HRTEM whereas the mutantpeptide SEQ ID NO:26, containing a polyglycine tail HWKHPSGAWDTLGGG [SEQID NO:32] and a control peptide SEQ ID NO:28 did not disperse thenanotube at the same concentration. The results are shown in FIG. 2.Panel A of FIG. 2 is an electron micrograph of nanotubes ropes untreatedwith any peptide. Panel B of FIG. 2 is an electron micrograph of singlewalled nanotubes after treatment with the carbon nanotube bindingpeptide of SEQ ID NO:13 showing disbursement of the nanotubes. Panel Cof FIG. 2 is an electron micrograph of single walled nanotubes aftertreatment with the peptide of SEQ ID NO:28, a control peptide havinglittle or no nanotube binding affinity.

Example 5 Graphite-Cleaned Binding Peptides

In order to find peptides with specific binding to carbon nanotubes,phage display experiments were performed as described in Example 2 onCNT-7 carbon nanotube substrates using a “graphite-cleaned” phagelibrary. The graphite-cleaned phage library was generated by firstwashing the complete phage library on a pyrolytic graphite substrate.The washed or cleaned library was thus denuded of phage that would bindto Graphite. Highly ordered pyrolytic Graphite (HOPG SPI-2, SPISupplies, West Chester, Pa.) was attached to a petri dish and a freshlayer of graphite was exposed using a Scotch tape. About 10¹¹ pfu M13phage in TBS-0.1% Tween-20® was added to the graphite substrate andallowed to sit for binding for 45-60 minutes at room temperature.Unbound phages were washed away with excess amount of (TBS-T) at definedconcentrations of Tween-20®. Bound phages were eluted with Glycine-HClbuffer at pH 2.3. The unbound phage (graphite-cleaned library) were thenused to perform phage display experiments on CNT-7 as described inExample 2. Individual phages were isolated and DNA sequences wereobtained using standard molecular biology methods described above.

After four rounds of phage display on CNT-7 with the graphite-cleanedlibrary (round 4 with 0.5% concentration of Tween-20®), two consensussequences emerged. These are:

HHHHLRHPFWTH (SEQ ID NO: 23) and WPHHPHAAHTIR (SEQ ID NO: 24)

The implication is that the binding of these sequences is specific tothe CNT-7, as compared to a graphitic clone. The significance of thefinding is in the close relationship between the graphene sheet thatbounds freshly cleaved graphite, and the surface of carbon nanotubes.Carbon nanotube surfaces are essentially curved or graphene sheets. Assuch, objects may bind both to carbon nanotubes and to graphite.Additional significance may be attached to those whose bindingdiscriminates between the two. This result illustrates that peptides canrecognize different allotropes of carbon.

Example 6 Peptide Facilitated Binding of CNT to Microspheres

Example 6 illustrates that microspheres coated with CNT binding peptidesare effective in binding single walled nanotubes and forcing assembly ofthe microspheres.

Preparation of Phage-Coated Microspheres.

Purified phage clones were amplified. Anti-mouse antibody IgG-coatedmicrospheres (seven microns in diameter from Bangs Laboratories, Inc,9025 Technology Drive, Fishers Ind. 46038-2886) were coated with ananti-M13 monoclonal antibody (Amersham pharmacia biotech Inc. 800Centennial Avenue PO Box 1327 Piscataway N.J. 08855). Purified phageclones were coated onto these microspheres in TBS buffer. Thephage-coated microspheres were incubated overnight with 10% Triton-X-165dispersed SWNTs 7.5 μg/ml in a dialysis tube against 1 L of TBS buffercontaining 10 grams of Amberlite XAD-4 (Sigma, P.O. Box 14508 ST. Louis,Mo. 63178). The microspheres were then washed three times with water.The beads were examined under SEM.

Preparation of Peptide-Coated Microspheres.

Selected sequences were synthesized as free peptides, including,NH₂-HWKHPWGAWDTLGGG-COOH (SEQ ID NO:25) and NH₂-HWKHPWGAWDTL-COOH (SEQID NO:13). Amino-modified microspheres (0.66 microns in diameter fromBangs Laboratories, Inc, 9025 Technology Drive, Fishers Ind. 46038-2886)were cross-linked to synthetic peptides at the C terminus through an EDClinker (Pierce Inc. 3747N. Meridian Road, P.O. Box 117, Rockford, Ill.61105 Sigma, P.O. Box 14508 ST. Louis, Mo. 63178). The peptide-coatedmicrospheres were incubated overnight with 10% Triton-X-165 dispersedSWNT 7.5 μg/ml in a dialysis tube against 1 L of TBS buffer containing10 grams of Amberlite XAD-4. The microspheres were then washed threetimes with TBS buffer. The beads were examined under SEM.

Results of contacting CNT's with microspheres either coated with CNTbinding phage or isolated peptide are illustrated in FIG. 3 a-d. FIG. 3(a) shows the surface of a microsphere coated with a control phage cloneexpressing peptide sequence NH₂-IDVESYKGTSMP-COOH. (SEQ ID NO:34).Clearly, there is no association of the carbon nanotubes with thissurface. FIG. 3( b) shows the surface of a microsphere coated with thebinding phage clone sequence NH₂-HWKHPWGAWDTL-COOH (SEQ ID NO:13). Itdemonstrates strong association between the phage and nanotube bundles.Similar results have been obtained with other nanotube-binding phageclones. FIG. 3( c), coated with the control peptideNH₂-LPPSNASVADYSGGG-COOH (SEQ ID NO:28), shows no association ofmicrospheres with nanotubes. Indeed, the suspension of microspheresremained highly dispersed. FIG. 3( d) shows strong association betweenthe microspheres coated with the binding peptide NH₂-HWKHPWGAWDTL-COOH(SEQ ID NO:13) and nanotubes. Essentially, the nanotubes cross-linkedthe microspheres, resulting in a loss of dispersion of the microspheresand formation of large clusters of microspheres.

Example 7 CMBP Generated to a Variety of Carbon Nanotube Substrates

This example illustrates that carbon nanotube binding peptides may begenerated to a variety of carbon nanotube substrates including thosemade by the HiPCo process and those that have undergone various cleaningprocesses.

A series of experiments to select carbon nanotube binding peptides wereperformed as described in Example 2. The first substrate used was SWNTsfrom CNI prepared using the HiPCo process that was prepared only by acidcleaning, dispersion in toluene and drying to form a mat. Peptidesresulting after the selection process are listed in SEQ. ID Nos:39-85.

The second substrate used in the selection process as described inExample 2 were MWNTs obtained from Yet-Ming Chiang, Department ofMaterials Science, MIT, Cambridge, Mass. Peptides that were selected arelisted in SEQ. ID Nos:1-4,6,8,10-13, 15, 16, 18-22, 28, 36, 92, 114-147,and 177. The above example was repeated used fresh MWNTs obtained fromthe same source. Resulting peptides are listed in SEQ. ID Nos:94-113 and177.

Graphite-cleaned MWNTs obtained from Yet-Ming Chiang, Department ofMaterials Science, MIT, Cambridge, Mass. prepared as described inExample 5 and were also used as substrates. Resulting peptides arelisted in SEQ. ID Nos:148-176.

1. A composition comprising a solid substrate coated with a peptidehaving an amino acid sequence selected from the group consisting of SEQID Nos: 167-177.
 2. The composition according to claim 1 wherein thepeptide is affixed on a phage.
 3. The composition according to claim 1further comprising at least one carbon nanotube bound thereto.